The main therapeutic challenge in the treatment of acute myeloid leukemias (AML) is the development of strategies aimed at overcoming resistance to chemotherapy and in particular to treat relapsed patients successfully. The factors regulating the survival of resistant leukemic cells are largely unknown . This project is designed to identify roadblocks to apoptosis in resistant leukemia cells and target mechanisms of apoptotic resistance. We have analyzed the expression of pro- and anti-apoptotic genes in newly diagnosed and relapsed AML and determined critical roles for Bcl-2, Mcl-1 and XIAP. Furthermore, we identified the microenvironment as important for chemosensitivity of AML and identified the chemokine receptor CXCR4 as a critical mediator. Finally,we analyzed PI3K/AKT/mTOR and Raf/Mek/Erk signaling in AML and established their functional and prognostic importance. These findings have led to the development of novel therapeutic concepts for AML. Molecules critical for AML survival became therapeutic targets and clinical trials have been initiated as a result (Proj.4,5). These clinical trials become the ultimate test to validate the hypotheses developed. First, we will test the hypothesis that activation of the intrinsic apoptosis pathway sensitizes AML and AML stem cells (LSC) to chemotherapy. Small molecules and genetic approaches will be used to target Bcl-2 and Mcl-1 , but the entire Bcl-2 family will be analyzed in bulk AML and LSC and correlated with response utilizing reverse-phase protein arrays (RPPA). The effects of kinases on Bcl-2 and Mcl-1 phosphorylation status and drug sensitivity will be determined. Second, we will explore p53 activation as a therapeutic strategy for AML, alone and in the context of Bcl-2 inhibition. We have already demonstrated that MDM2-inhibition alone induces p53 signaling and apoptosis in AML with wtp53. We will now investigate the role lof MDMX, of p53 acetylation and phosphorylation and interactions of p53 and Bcl-2 to better understand the observed activity of MDM-2 inhibitors and their synergism with Bcl-2 inhibitors. Third, we will develop in vitro models of bone marrow microenvironment, analyze signaling pathways induced in leukemias and stroma, and investigate effects of CXCR4, VLA-4 and ILK inhibitors in overcoming microenvironment-mediated drug resistance. If successful, these studies will provide rationale for greatly improved therapies for AML that target both, the leukemic cells and their microenvironment.